Tertiary butylhydroquinone alleviated liver steatosis and increased cell survival via β-arrestin-2/PI3K/AKT pathway

Objective(s): This study aimed to evaluate the effects and the underlying mechanisms of tertiary butylhydroquinone (TBHQ) on diabetic liver steatosis and cell survival. Materials and Methods: We performed streptozocin injection and used a high-sugar-high-fat diet for mice to develop an animal model of type 2 diabetes mellitus (T2DM). Bodyweight, blood glucose levels, and content of insulin were measured on all of the mice. The liver tissues were observed by hematoxylin-eosin staining. Protein levels of the liver were measured by Western blot analysis in mice. Primary hepatocytes were induced by hypochlorous acid (HClO) and insulin to form insulin resistance (IR). Primary hepatocyte apoptosis was observed by Hoechst staining. The PI3K/AKT signaling pathway and β-arrestin-2 factor were evaluated by Western blot assay. Results: TBHQ reduced the blood glucose level and content of insulin in serum, increased body weight, and effectively alleviated liver steatosis in diabetic mice. TBHQ significantly up-regulated the expression of p-PI3K, p-AKT, GLUT4, GSK3β, and β-arrestin-2 in the liver of diabetic mice. Cell experiments confirmed that TBHQ increased the survival ability of primary hepatocytes, and TBHQ improved the expression of p-PI3K, p-AKT, GLUT4, and GSK3β by activating β-arrestin-2 in primary hepatocytes. Conclusion: TBHQ could alleviate liver steatosis and increase cell survival, and the mechanism is due in part to β-arrestin-2 activation.


Introduction
Insulin resistance (IR) is an important cause for the development of type 2 diabetes mellitus (T2DM) (1)(2)(3)(4), which is a disease associated with abnormal metabolic function and will lead to abnormally high levels of blood glucose (5,6). The long-term existence of hyperglycemia in diabetes leads to liver steatosis. Therefore, ameliorated IR plays a central role in treating liver steatosis of T2DM.
Tertiary butylhydroquinone (TBHQ) is the most commonly used synthetic antioxidant and can enhance the effect of insulin in vivo (7,8). TBHQ remarkably increases PI3-kinase-dependent Akt (also referred to as PKB) phosphorylation (p-Akt) (9,10), which could reflect insulin sensitivity in hepatocytes and nerve cells (11,12). The PI3K/Akt signaling pathway plays a vital role in regulating cell apoptosis and glucose metabolism (13). The activation of p110 and p85 subunits of PI3K can catalyze Akt phosphorylation, then lead to positive regulation of the PI3K/Akt signaling pathway (14,15). In addition, p-Akt stimulates the glucose metabolism via controlling the insulin-stimulated translocation of GLUT4 that plays an important role in insulin action (16) and controlling the activity of glycogen synthesis kinase-3β (GSK-3β) that has an important effect on glycogen synthase, thereby promoting glucose uptake, glycogen synthesis, insulin sensitivity (17,18) and regulation of the cell cycle (19,20). However, the underlying mechanism of TBHQ increases p-Akt thus enhances insulin effect, and hepatocytes apoptosis is unclear.
The aim of this study, therefore, was to establish a link between β-arrestin-2 and PI3K/Akt signaling pathwayrelated hepatocyte apoptosis and liver steatosis, then to determine if TBHQ could inhibit these pathological processes by activating the pathway. The work described here demonstrates that exposure of hepatocytes to TBHQ increased β-arrestin-2 expression, promoted PI3K/AKT pathway, blocked hepatocytes apoptosiss, and liver steatosis.

Protocols for animal experiments
Four-week-old male ApoE-/-mice were purchased from Model Animal Research Center GemPharmatech Co., Ltd. of Nanjing University (Qualified number: 201804855). These animal studies were approved by the Xinxiang Medical University Veterinary Medicine Animal Care and Use Committee. The mice were housed in a temperature and humidity-controlled room on a light/dark cycle of 12 hr. Low-dose of STZ and highsucrose-high-fat diet has been used to induce the mice model of T2DM.
The mice were randomly divided into 5 groups (n=10 for each group) after one week of acclimation period: The control group, mice fed a conventional diet; The control+TBHQ group, mice fed a conventional diet and gavaged with TBHQ at a dose of 60 mg/kg/day (36,37); The T2DM group, mice fed a high-sucrose-high-fat diet; The T2DM+TBHQ group, mice fed a high-sucrose-highfat diet and gavaged with TBHQ at a dose of 60 mg/kg/ day; The T2DM+rosiglitazone group, mice fed a highsucrose-high-fat diet and gavaged with rosiglitazone at a dose of 7 mg/kg/day (38,39). The T2DM group, the T2DM+TBHQ group, and the T2DM+rosiglitazone group of mice were subjected to an injection of STZ (50 mg/kg body weight in 10 mM citrate buffer, pH 4.0, IP) after a 10 hr fast (40) and one week later, the T2DM+TBHQ group and the T2DM+rosiglitazone group began receiving a high-sucrose-high-fat diet and were gavaged with TBHQ and rosiglitazone.
All mice were given a high-sucrose-high-fat diet (sucrose: lard: sodium cholate: cholesterol: conventional feed=20: 10: 7: 3: 60) except for the control group and control+TBHQ group during the entire treatment period (41). The protocol of the animal experiment was shown in Figure 1A.

Biochemical study
The body weight and blood glucose levels of all mice were measured each week during the experimental period. Blood was collected from the mice eye socket vein and centrifuged at 4000 rpm for 15 min at 4 °C to isolate serum. Serum and liver samples were stored in a -80 °C refrigerator for further biochemical assessment (42). All animals' liver tissues were collected.

Assay of serum insulin
Serum insulin levels of mice were assayed using an ELISA kit following the manufacturer's instructions.

Hematoxylin-eosin (HE) staining
Liver tissues fixed in 4% PBS-buffered neutral formaldehyde for 48 hr were embedded in paraffin and cut into a section with 4 μm thickness, then stained by HE staining (43). Lesion area related to swollen hepatocytes, granular degeneration, steatosis, and lipid vacuolization in the liver, was contrast-adjusted with the Image J software (National Institutes of Health, Bethesda, MD, USA).

Apoptosis experiments
According to the manufacturer's instructions, Hoechst staining was performed using the Hoechst staining kit in primary hepatocytes. The apoptosis cells were counted using the Image J software (National Institutes of Health, Bethesda, MD, USA).

Western blot analysis
Protein levels were analyzed by Western blot analysis (44,45).

Statistical analysis
Statistical analysis was performed using SPSS software. All quantitative results data were expressed as mean±SEM. All results were performed by one-way ANOVA with Newman-Student-Keuls test for multiple comparisons, and for both sides P<0.05 were considered significant.

TBHQ obviously alleviated the abnormal glucose metabolism in diabetic mice
A high-sucrose-high-fat diet treatment and STZ injection-induced diabetes. The fasting blood glucose (FBG), postprandial blood glucose (PBG), and bodyweight levels of experimental mice are indices of diabetes. As shown in Figure 1B-D, data were examined over different time intervals of all experimental mice. FBG and PBG significantly decreased in comparison with diabetic mice after six weeks of rosiglitazone or TBHQ treatment. The bodyweight of the T2DM group obviously decreased during six weeks. However, the bodyweight of the T2DM group significantly increased with TBHQ or rosiglitazone treatment. We examined the serum insulin levels of all experimental groups, as shown in Figure 1E. IR was observed in the T2DM group as evident by increasing serum insulin levels compared with the control group. The serum insulin values obviously decreased in comparison with diabetic mice after six weeks of rosiglitazone or TBHQ treatment. These data demonstrate that the persistent application of TBHQ relieves glucose metabolic disorders and IR in diabetic mice induced by high-sucrose-high-fat diet treatment and STZ injection.

TBHQ effectively alleviated liver steatosis of diabetic mice and increased cell survival in primary hepatocytes
To directly assess whether TBHQ treatment has influences on T2DM, the lesion area in the liver was determined by HE staining (Figure 2A). As indicated in Figures 2A-B, Liver histopathological examination showed no abnormality of the hepatocyte architecture and morphology in normal mice. In contrast, highsucrose-high-fat diet treatment and STZ injectioninduced T2DM mice exhibited severe hepatocyte necrosis and macrovesicular steatosis in the hepatocytes. Whereas TBHQ feeding for six weeks exhibited a beneficial effect on diabetes-mediated pathological changes, the degeneration of the hepatocytes markedly alleviated, and macrovesicular steatosis and cell death partially recovered. These results demonstrate that the persistent application of TBHQ relieves hepatic function in diabetic mice.
Hoechst staining was used to explore the effect of TBHQ on primary hepatocyte apoptosis. The cells with apoptotic morphology appearing condensed or fragmented nuclei were counted. As shown in Figures  2C-D, cell treatment with HClO, TBHQ, and insulin displayed varying degrees of apoptosis, we found that HClO promotes cells apoptosis. The number of apoptosis cells markedly down-regulated after TBHQ treatment.
These results indicate that TBHQ subsequently increases the survival ability of primary hepatocytes.

Effects of TBHQ treatment on the PI3K/AKT pathway in diabetic mice and primary hepatocytes of insulin resistance
To confirm the regulatory effect of TBHQ on liver tissues, we next evaluated the glucose metabolismrelated proteins. The hepatic protein levels of GLUT4, GSK3β, p-PI3K, and p-AKT are the indices of diabetes ( Figures 3A, C, E, and G). We found that TBHQ increased protein expression of the glucose transport marker GLUT4 and glycogen synthase marker GSK3β when compared with the T2DM group. Moreover, the p-PI3K and p-AKT levels were relatively elevated in diabetic mice in comparison with T2DM mice. Many studies have shown that the protein expression level of p-AKT is up-regulated by the phosphorylation of PI3K (47). Therefore, the level of p-AKT in liver tissues was inspected to discuss whether TBHQ increased p-AKT activity by activating the PI3K pathway and then regulating gluconeogenesis. These results suggest that TBHQ may inhibit glucose uptake and increase glycogen synthesis via the PI3K/AKT pathway.
To further confirm the regulatory effect of TBHQ on suppressing glucose uptake and increase glycogen synthesis in the liver of diabetic mice via the PI3K/AKT pathway, we conducted a cell experiment and found that HClO and insulin treatment decreased protein levels of GLUT4, GSK3β, p-AKT, and p-PI3K in primary hepatocytes, which were effectively improved by TBHQ in comparison with cells of insulin group (Figures 3B,  D, F, H). Exposure of primary hepatocytes to TBHQ significantly enhanced the phosphorylation levels of AKT and PI3K in HClO and insulin-treated primary hepatocytes. Protein levels of p-PI3K, p-AKT, GLUT4, and GSK3β were relatively elevated in comparison with HClO and insulin-treated primary hepatocytes. Collectively, these results further demonstrate that TBHQ attenuates diabetes by inhibiting glucose uptake and increasing glycogen synthesis via the PI3K/AKT pathway.

TBHQ might activate β-arrestin-2 factor in diabetic mice and primary hepatocyte cells of insulin resistance
To examine whether β-arrestin-2 activation is necessary for TBHQ to ameliorate glucose metabolism, we detected β-arrestin-2 in our animal and cell experiments and found that the expression of β-arrestin-2 has significantly decreased in both diabetic mice liver and IR cells (Figures 4A-B). However, after TBHQ treatment, β-arrestin-2 was significantly increased, which has the same effect when compared with rosiglitazone. TBHQ can correct the abnormal glucose metabolism and increase the expression of β-arrestin-2, but whether TBHQ improves glucose metabolism via β-arrestin-2 still needs to be illuminated. We reduced the expression of β-arrestin-2 with β-arrestin-2-targeting siRNA in primary hepatocytes. Then we found that after TBHQ treatment, β-arrestin-2 in HClO+insulin-treated primary hepatocytes was relatively elevated in comparison with β-arrestin-2 down-regulated HClO+insulin-treated primary hepatocytes ( Figure 4C). Collectively, these data suggest that TBHQ can improve IR by activating the β-arrestin-2 factor.

TBHQ activated β-arrestin-2 factor to increase insulin sensitivity in HClO and insulin-treated primary hepatocytes
To validate our hypothesis that TBHQ relieves IR by activating the β-arrestin-2 factor, we used β-arrestin-2 siRNA to decrease β-arrestin-2 expression in primary hepatocytes. Then protein expression levels of p-AKT,  Figures 5A-D). Taken together, these results suggest that TBHQ activates the β-arrestin-2 factor to improve the protein expression of GLUT4, GSK3β, p-PI3K, and p-AKT in case of insulin resistance.

Discussion
Insulin-resistant induced liver steatosis and hepatocellular injury (48) are complications of T2DM (1-6). TBHQ, as a food antioxidant, can increase the effect of insulin, thus reducing IR via p-Akt (7-10), which plays a vital role in regulating glucose metabolism and cell apoptosis (13). However, whether TBHQ can alleviate liver steatosis and the mechanisms behind the antiinsulin resistance and anti-apoptosis effects of TBHQ has not been clearly defined. Our data presented here was  the first to show the effectiveness of TBHQ on improving liver steatosis in T2DM and the mechanism of the antiinsulin resistance and anti-apoptosis effects of TBHQ related to the β-arrestin-2/PI3K/AKT pathway. The data suggested that the persistent application of TBHQ could effectively reduce the levels of blood glucose, increase the body weight, alleviate liver steatosis and activate β-arrestin-2. Also, a parallel experiment in cells was conducted. In vitro studies suggested that TBHQ could increase the survival of HClO induced insulin-resistant primary hepatocytes and the mechanism related to the β-arrestin-2/PI3K/AKT pathway. In brief, TBHQ may potentially be used as a medicinal compound for the treatment of T2DM via activating the β-arrestin-2/ PI3K/AKT pathway. TBHQ, which has antioxidant and anti-inflammatory effects, can protect pancreatic islet cells from damage and increase insulin sensitivity in vivo to improve T2DM. Liver tissue, as a main detoxifying organ, is often damaged by metabolic overload, lipid accumulation, and viruses' factors. Moreover, liver tissue metabolic disturbance will occur in T2DM. TBHQ can inhibit liver cell apoptosis, which protects the liver from acute and chronic toxin-mediated injury (35,49,50). According to these pieces of evidence, we hypothesized that TBHQ can alleviate liver injury in T2DM, and our subsequent experiment supports this point.
β-Arrestin-2 is a key component in the regulation of insulin sensitivity (15), and the lack of β-arrestin-2 can inhibit the resistibility of insulin in glucose uptake, which leads to abnormal glucose tolerance in vivo (51). TBHQ can activate β-arrestin-2 and promote the autophagy of hepatocytes, thus exerting the effect of anti-fatty acid (25). In our experiments, we detected the expression of β-arrestin-2 both in in vivo and in vitro studies. Consistently, we found that β-arrestin-2 expression has decreased in both diabetic mice liver and IR cells. However, after TBHQ treatment, the expression of β-arrestin-2 has significantly increased. In order to further explore the underlying mechanism, we used β-arrestin-2 siRNA in primary hepatocytes and found that β-arrestin-2 in HClO+insulin-treated primary hepatocytes was relatively elevated in comparison with β-arrestin-2 down-regulated HClO+insulin-treated primary hepatocytes after TBHQ treatment, which suggested that TBHQ alleviated insulin-resistant directly via β-arrestin-2. Previous reports have suggested that insulin can tremendously decrease blood glucose by improving phosphorylates AKT and PI3K, which is associated with GSK3β and GLUT4 (52)(53)(54). β-arrestin-2 can induce AKT activation by phosphorylating two major residues: Ser473 and Thr308 (25). Thus, we guessed that TBHQ alleviates IR and liver steatosis via activating β-arrestin-2/PI3K/AKT pathway. Our results presented that TBHQ could enhance the phosphorylation levels of AKT and PI3K, thus significantly improving the role of GLUT4 and GSK3β in the liver of diabetic mice and primary hepatocytes of IR. After β-arrestin-2 knockdown, the PI3K/AKT pathway was significantly influenced.
Rosiglitazone, a thiazolidinedione antidiabetic agent, improves IR (55). In animal models of IR, rosiglitazone decreased plasma glucose, insulin, and triglyceride levels and also attenuated or prevented diabetic nephropathy and pancreatic islet cell degeneration (55).
Some studies suggested that rosiglitazone promotes glucose metabolism of GIFT tilapia based on the PI3K/ Akt signaling pathway (56), which may have the same effect as TBHQ in treating T2DM. Clinical analysis has suggested that it should be used with caution in patients with cardiac insufficiency, severe cardiovascular disease, and hypertension (57). It also may increase the occurrence of cardiovascular complications in the treatment of T2DM. Therefore, developing highefficiency and safe drugs that can prevent diabetes is very necessary. TBHQ is mainly used as a food additive. TBHQ is safe and low toxicity for the human body, and its chemical structure is modified to enhance the antioxidant effect. In our study, we found that TBHQ can improve IR except for the antioxidant effect . Thus TBHQ is expected to be the ideal medicine for treating T2DM.
As we all know, diabetes can cause damage to the liver, skeletal muscles, and fat. The shortcoming of this study is that we have only explored the effect of TBHQ in relieving liver damage, we have not probed the role of TBHQ in improving injury of skeletal muscles and fat.
In the next study, we will use C2C12 and 3T3-L1 cells to research the effect of TBHQ in these diseased organs. As different tissues have different sensitivities to TBHQ, we will use multiple doses of TBHQ to explore the optimal dose of TBHQ in diabetes. Only in this way can we make TBHQ an ideal medicine and use it as soon as possible for treating T2DM patients.

Conclusion
In summary, this study demonstrates that TBHQ alleviates T2DM via β-arrestin-2 activation and β-arrestin-2 mediates liver steatosis and cell apoptosis via PI3K/AKT signaling pathway ( Figure 6). Therefore, TBHQ can be a potential medicine and β-arrestin-2 is a pharmacological target for the therapy of T2DM.

Acknowlegment
The results presented in this paper were part of a

Availability of Data and Material
The data supporting the findings can be found in supplementary materials.